Project Summary: Cutaneous squamous cell carcinoma (cSCC) is the second most common form of cancer with approximately 700,000 cases annually in the US leading to 3,000 deaths. The incidence of cSCC will increase given an aging population with rising cumulative UV exposure. In response to this epidemic, the Surgeon General issued a `Call to Action' detailing a plan to address the growing epidemic of UV-induced skin cancers. One component of this strategic plan to decrease UV-induced skin cancer is to perform research that defines the mechanisms of UV-induced skin cancer. The transition from keratinocyte to cSCC by UV radiation involves formation of precancerous lesions, called actinic keratoses (AKs) and carcinoma in situ (SCCIS). AKs and SCCIS are hypothesized to arise from UV-induced mutations in keratinocytes that lead to cell populations with aberrant growth and differentiation. To better understand how UV irradiation alters keratinocytes and promotes the early stages of skin cancer, we performed laser capture microdissection of SCCIS and adjacent UV-exposed epidermis, isolated genomic DNA and generated libraries for whole exome sequencing. This novel approach permits a precise comparative genomic analysis of mutations in UV-exposed epidermis and SCCIS. Our exomic sequencing data demonstrate low frequency UV-signature loss-of-function mutations in Notch in 60% of epidermal samples and heterozygous clonal mutations in 40% of SCCIS samples. Nucleoporins (Nups) were also frequently mutated in 80% of epidermal libraries and 100% of SCCIS libraries. These data show that UV-signature mutations in Notch and Nups are more common than mutations in known oncogenes such as p53 or RAS. The goal of this proposal is to show how Notch and Nup mutations promote the early stages of UV-induced skin carcinogenesis leading to SCCIS. The biological impact of UV- irradiation on Notch-deficient clones will be studied using genetically engineered mice and engineered human keratinocytes in xenograft models. The biological significance of Nup mutations in UV-irradiated skin will be determined using mice with an epidermal deficiency in the Nup Elys, which links the nuclear pore complex to chromatin. In addition, human keratinocytes will be engineered to express mutant Nups and subjected to UV-irradiation in a xenograft model. The aims of this proposal will yield novel insights into the role of Notch and Nup mutations in skin cancer and will provide important insights into designing rational approaches to treat SCCIS.